The present invention provides novel substituted pyrimidines. These compounds have been found to inhibit phospholipase A2 activity, in particular cPLA2 (cytosolic phospholipase A2).
Phospholipases A2 (PLA2s; EC 3.1.1.4) are enzymes that hydrolyze the 2-acyl ester bond of phosphoglycerides generating free fatty acids and lysophospholipids (for review, see, Kramer, R M (1993) Advances in Second Messenger and Phosphoprotein Research 28: 81; Glaser et al. (1993) TiPS 14: 92; Dennis E A (1994) J. Biol. Chem. 269: 13057). PLA2s are a diverse class of enzymes with regard to function, localization, regulation, mechanism, sequence, structure, and role of divalent metal ions. A variety of polypeptide species can exhibit PLA2 activity; for purposes of this specification, these polypeptides are considered PLA2 isozymes.
In general, PLA2 enzymes catalyze the hydrolysis of the fatty acid ester bond at the sn-2 position of membrane phospholipids to produce arachidonic acid and its metabolites. Group I, II, and III PLA2s are extracellular enzymes of approximately 14-18 kD in humans, and are designated sPLA2s, in recognition of their secretion. sPLA2s are found in many extracellular fluids and have a broad substrate specificity for many types of phospholipids.
Group IV PLA2 is a cytosolic enzyme of approximately 85 kD (based on deduced cDNA coding sequence) to 110 kD (based on SDS-PAGE of purified protein), and is designated cPLA2 to indicate its cytosolic location. Unlike sPLA2s, the cPLA2 enzyme exhibits preferential catalysis of phospholipids which contain arachidonic acid, and is most likely the enzyme responsible for arachidonic acid release which is the rate-limiting step for subsequent eicosanoid biosynthesis of pro-inflammatory lipid mediators (prostaglandins, leukotrienes, lipoxins, and platelet-activating factor: xe2x80x9cPAFxe2x80x9d). cPLA2 is present in the cytosol of a variety of species and cell types, including human U937 cells (monocytes), platelets, kidney, and macrophages, among others, and is implicated in controlling arachidonic acid metabolism and eicosanoid production.
Some cells contain calcium independent phospholipase A2/B enzyme. The phospholipase A2/B purified enzyme is characterized by activity in the absence of calcium and having a molecular weight of 86 kD on SDS-PAGE (see, U.S. Pat. Nos. 5,554,511 and 5,466,595).
Of particular interest in the present invention is the cPLA2 enzyme. Human cPLA2 has been cloned as a cDNA isolated from mRNA of a human monocytic cell line, (U.S. Pat. Nos. 5,354,677 and 5,328,842; Sharp et al. (1991) J. Biol. Chem. 266: 14850; Clark et al. (1991) Cell 65: 1043) and the mRNA encodes a protein of 749 amino acids which has little detectable homology with the secreted sPLA2s or any other protein in known sequence databases. The cPLA2 cDNA identifies a single copy gene in the human genome, with no detectable closely related genes based on Southern blotting experiments. A suitable source of cPLA2 can be obtained, if desired, by expression of a recombinant expression vector in a suitable host cell, as described in U.S. Pat. No. 5,354,677, or by conventional biochemical purification from mammalian cells, as is known in the art.
Moreover, cPLA2 contains an amino-terminal domain which binds calcium and similar divalent cations, and cPLA2 binds to membrane vesicles at submicromolar concentrations of Ca+2 in a calcium-dependent fashion. cPLA2 can translocate to membranes when activated in the presence of calcium. Presumably, cPLA2 associates with membrane components in vivo under suitable calcium concentrations. Agents that stimulate the release of arachidonic acid (ATP, thrombin, phorbol ester, calcium ionophore) can cause increased serine phosphorylation of cPLA2 which increases the enzymatic activity of cPLA2 (Lin et al. (1993) Cell 72: 269). Phosphorylation is believed to contribute to the control of cPLA2 activity in vivo (Lin et al. (1992) Proc. Natl. Acad. Sci. (USA) 89: 6147; Lin et al. (1993) Cell 72: 269; Qiu et al. (1993) J. Biol. Chem. 268: 24506; Kramer et al. (1993) J Biol. Chem. 268: 26796).
The art generally recognizes the physiologic role of cPLA2 to be in the mediation of inflammation via its role in arachidonic acid metabolism and lipid/lipoprotein metabolism, such as cell membrane homeostasis. Roshak et al. (1994) J. Biol. Chem. 269: 25999 used antisense oligonucleotides complementary to the cPLA2 mRNA to inhibit prostaglandin production in LPS-induced monocytes, indicating a potential role for cPLA2 in generating inflammatory regulators in monocytes. Verity MA (1993) Ann. N.Y. Acad. Sci. 679:110 speculates that xe2x80x9cabusive activationxe2x80x9d of PLA2 via uncontrolled Ca+2 influx might produce irreversible cell injury of neurons via extensive localized lipid peroxidation and subsequent membrane disintegration. U.S. Pat. Nos. 5,354,677 and 5,328,842 indicates that cPLA2 inhibitors are expected to be used to treat inflammatory conditions, such as psoriasis, asthma, and arthritis (see, col. 15), and prophesizes that such anti-inflammatory compounds can be identified as cPLA2 inhibitors.
In addition to the roles mentioned above, PLA2 activity has been implicated as a contributor to destructive cellular processes in various tissues including, but not limited to, the central nervous system. PLA2 activity has also been reportedly involved in ischemic injury and pathological nervous system conditions.
A number of inhibitors of PLA2 activity have been reported. Bromoenol lactone and trifluoromethyl ketones (e.g., palmitoyl trifluoromethyl ketone, arachidonyl trifluoromethyl ketone) have been reported to be capable of inhibiting a Ca+2-independent PLA2 activity (Ackermann et al. (1995) J. Biol. Chem. 270: 445) as well as cPLA2 (Street et al. (1993) Biochemistry 32: 5935). Several benzenesulfonamide derivatives have also been reported to be capable of inhibiting PLA2 activity (European Patent Application 468 054; Oinuma et al. (1991) J. Med. Chem. 34: 2260).
In view of the role PLA2 can play in destructive cellular processes, there is a need in the art for new compounds that are inhibitors of PLA2. These compounds can then be used to treat or prevent PLA2-mediated diseases. The present invention provides such new compounds, compositions and methods of treatment.
The present invention provides substituted pyrimidines which are effective inhibitors of PLA2, more particularly cPLA2. As such, the present invention provides novel substituted pyrimidines which have the general structure: 
with the symbols R1, R2, R3, R4 and R5 representing the groups provided in the detailed description below. These compounds have activity as inhibitors of phospholipase A2, and in particular cytosolic phospholipase A2.
In other aspects, this invention provides pharmaceutical compositions of the substituted pyrimidine compounds of formula I or a pharmaceutically acceptable salt thereof with a pharmaceutically acceptable carrier.
As described in detail below, the compounds of formula I are useful in the treatment of conditions associated with PLA2-mediated conditions, such as inflammation and Alzheimer""s disease. As such, the present invention provides a method of treating cPLA2-dependent diseases. In this aspect, the method comprising administering to a subject having at least one of cPLA2-mediated disease with an effective amount of a compound of formula I.
The compounds and pharmaceutical compositions of compounds of formula I are also useful for in vitro assays for PLA2 inhibitors. As such, in another aspect, the present invention provides a method of inhibiting PLA2 activity in vitro, comprising contacting a cell having PLA2 activity with a compound of formula I and assaying the PLA2 activity.
In another embodiment, the present invention provides the use for the manufacture of a medicament for the treatment of inhibiting PLA2 activity and treating cPLA2-dependent diseases. These and other aspects of the present invention will be described in detail hereinbelow.